Many members of the Enterobacteriaceae such as Shigella and Escherichia coli are known to produce one or more toxins. Amongst these are several potent cytotoxins and neurotoxins. Shigella dysenteriae is known to produce the so-called Shiga-toxin (Sandvig, K., Toxicon 39: 1629-1635 (2001)). A group of very closely related Escherichia coli toxins is toxic to African green monkey (vero) cells, and thus they became known as verotoxins. These toxins show a close resemblance to a cytotoxic toxin that was earlier found in Shigella dysenteriae type 1, which explains their currently used name: Shiga-like toxins (SLT). The Shiga-like toxins have been described i.a. in a review by Agbodaze, D. (Comp. Immunol., Microbiol. & infectious diseases 22: 221-230 (1999)) and in a review by O'Brian, D. and Holmes, R. K. (Microbiol. Review 51: 206-220 (1987)).
It goes without saying that the invention is applicable to both the Shiga-toxin and the Shiga-like toxins. Shiga-like toxins are now known to be the cause of i.a. hemorrhagic colitis and hemolytic-uremic syndrome in humans (Karmali et al., Lancet I: 1299-1300 (1983)), diarrhea in calves (Chanter, N., Vet. Microbiol. 12: 241-253 (1986) and Mainil et al., Am. J. Vet. Res. 48: 734-748 (1987)) and edema disease in swine (Dobrescu, L., Am. J. Vet. Res. 44: 31-34 (1983), Gannon, V. P. J. at al., Can. J. Vet. Res. 53: 306-312 (1989), Marques, L. R. M., et al., FEMS Microbiol. Letters 44: 33-38 (1987), Smith, H. W. et al., J. Gen. Microbiol. 129: 3121-3137 (1983) and Smith, H. W. et al., J. Med. Microbiol. 1: 45-59 (1968)).
Clinical manifestations of edema in pigs, i.a. neurological dysfunction, result from microangiopathy and vascular necrosis caused by a specific Shiga-like toxin variant Stx2e (Neilsen, N, O., Edema Disease, p. 528-540 (1986) In A. D. Lehman, Straw, B., Glock R. D. et al. (ed.), Diseases of swine, 6th ed. Iowa State University Press, Ames. USA), (Gannon, V. P. J. at al., Can. J. Vet. Res. 53: 306-312 (1989), Kurtz, H. J. et al., Am. J. Vet. Res. 30: 791-806 (1969) and Marques, L. R. M., et al., FEMS Microbiol. Letters 44: 33-38 (1987)). This variant Stx2e, also known in the art as SLT-IIe, SLT-IIv, Verocytotoxin 2e and VT2e, causes a disease that strikes approximately one week following weaning. The disease, characterised by the edema and the subsequent specific neurological disturbances that it causes, is generally known as post-weaning edema (PWE) or edema disease.
Shiga-toxin and all Shiga-like toxins share the same general structure. They consist of a single A-subunit bound to multiple copies of a B-subunit. Normally, a single A-subunit is bound to a pentamer of B-subunits. The A-subunit is the actual toxin-part: it plays a role in the inhibition of the host's protein synthesis. The B-subunit, more specifically when in its pentamer form, is associated with receptor binding. A single B-subunit is about 7.5 kD, whereas the A-subunit is about 32 kD.
The DNA-sequence of the A1-part (see below) of the Shiga-like toxin variant Stx2e is provided in SEQ ID NO: 1.
FIG. 1: shows a schematic drawing of a typical Shiga-like toxin; its overall structure, the location of the A1/2 parts (see below) of the A-subunit and the location of the B-subunits.
The whole toxin is therefore best described as a bipartite toxin (i.e.: a toxin consisting of two parts) comprising a single A-subunit and single pentamer formed by 5 B-subunits. The A-subunit as such can subsequently be functionally divided into an A1-part being the actual enzymatic part, and an A2-part being the part of the A-subunit involved in binding to the pentamer of B-subunits. The binding of the A-subunit, through the A2-part of the A-subunit, to the B-subunit follows the lock-and-key principle: the A2-part of the A-subunit of Shiga-like toxin only fits into the B-subunit of Shiga-like toxin, and not to other, though closely related, B-subunits such as e.g. the B-subunit of the Heat-labile enterotoxin (LT) of Escherichia coli. 
It is known that vaccination with inactivated toxins can be used to prevent disease caused by Shiga-like toxin producing E. coli strains. (Awad-Masalmeh, M., In Proc of the 10th Int. Pig Vet. Soc. Congress, R10 de Janeiro, Brazil (1988), Awad-Masalmeh, M., Dtsch. Tieraertzl. Wochenschr. 96: 419-421 (1989), Howard, J. G., Br. J. Exp. Pathol. 36: 439-4476 (1955), Islam, M. S., and Stimson, W. H., J. Clin. Lab. Immunol. 33: 11-16 (1990), MacLeod, D. L and Gyles, D. L., Vet. Microbiol. 29: 309-318 (1991), Wadolkowsky, E. A. et al., Infect. & Immun. 58: 3959-3965 (1990), Bosworth, B. T. Infect. & Immun. 64: 55-60 (1996)).
The genomic organisation as well as the location and sequence of the genes encoding the A- and B-subunits for Shiga-like toxins is known (Spicer E. K. et al., J. Biol. Chem, 257:5716-5721 (1982), Calderwood, S. B. et al., Proc. Natl. Acad. Sci. USA 84: 4364-4368 (1987), Dallas W. S. and Falkow S., Nature 288: 499-501 (1980), Leong J. et al., Infect. Immun. 48: 73-77 (1985)).
Therefore, in principle, having the necessary genetic information at hand, and knowing that vaccination with inactivated toxins can be used to prevent disease caused by Shiga-like toxin producing E. coli strains, large-scale in vitro expression of the genes encoding the A- and B-subunits seems a good starting point for vaccine production.
Against expectations however, although very efficient for the production and subsequent purification of both the A- and B-subunit of the comparable Heat-labile enterotoxin (LT) of Escherichia coli (see below), expression/purification turned out to be very difficult for Shiga-toxin and Shiga-like toxins.
First of all, although expression of the Shiga-like toxin B-subunit in a bacterial expression system is not a problem (Acheson et al., Infect. & Immun. 63: 301-308 (1995)), the Shiga-like toxin A-subunit can not, or only in minute quantities be expressed in bacterial expression systems.
Moreover, purification of the bipartite Shiga-toxin and Shiga-like toxin (contrary to the purification of LT) is both difficult and expensive. PCT-patent application WO 98/54215 provides ways of overcoming the difficulties experienced with purification, but relies therefore upon the use of affinity columns using expensive affinity ligands comprising disaccharides. For the preparation of a Shiga-toxin or Shiga-like toxin-based vaccine, this method of purification is from an economical point of view less desirable.
Therefore, both the expression and the purification of a Shiga-toxin or Shiga-like toxin remain problematic.